Organ transplantation is the treatment of choice for most patients suffering end stage diseases of the kidney, pancreas, liver, heart or lung. However, many patients suffer from unreconstructable tissue loss secondary to burns, traumatic injuries, immunological diseases, congenital anomalies or tumor resections. To date, these patients have been required to adapt to their deficiency, and ?in the case of amputees- utilize prostheses that, while helpful, remain not sensitive, limited in functionality, and cosmetically sub-optimal. Composite Tissue Allotransplantation (CTA) is the transplantation of multiple tissues (skin, muscle, bone, joint, cartilage, nerve, tendon, vessel) as a functional unit. CTA can be applied to reconstruct any tissue with cadaver donor tissue from a similar tissue source. CTA has emerged as an amalgamation of advanced microsurgical techniques for limb and flap autotransplantation, and improved immunosuppressive agents to prevent rejection. However, as a developing field, it has yet to have its unique immunological properties established. In a survey of biopsy specimens from human limb transplant cases, we have shown that vasculitis, dermatitis, myositis and perineural involvement can all be seen during human CTA rejection. However, given the limited availability of human tissue, the progression of the immune response remains relatively uncharacterized. Thus, animal models are required for the systematic evaluation of all CTA tissues. Currently there is not a non-human primate model (NHP) to investigate CTA. Therefore, the aim of our studies is to establish a means for pre-clinical, non-human primate testing of promising new approaches to composite tissue allotransplantation (CTA) that are being considered for use in humans, and to determine if these approaches are sufficiently safe and efficacious to warrant clinical experimentation. It is fundamental to establish an ethical and biologically relevant model of CTA in non-human primates. In particular, the transplantation of digits, hands and/or limbs in a non-human primate may significantly impair their social activities leading to problems in their development. Conversely, the model we propose will leave no functional deficit in the animal even in the case of removal. Currently, methods of immunosuppression are inadequately efficacious of prohibitely toxic to allow for a broad use in CTA. Therefore, once the model is established, an adequate immunosuppresive regimen will be studied. The model we propose to develop involve the transfer of bone, muscle, nerve, artery, vein and skin as a unit in monkeys. The effect of current immunosuppression on the time to rejection of the different tissues and on the character of that rejection is assessed after having established the normal healing process of a composite tissue transfer. We will use the CTA model to test therapies and the biology of the different tissues transplanted as a unit that have already been shown to be very promising in rodent models. The monkey composite tissue allotransplant model is one of the most accurate reflections of the way humans respond to transplanted tissues and the medications given to prevent rejection. Results. We utilized a sensate osteomyocutaneous radial forearm flap that avoids functional impairment even in the case of graft loss. The model was evaluated in monkeys that underwent auto- or allo-transplantation, with or without sub-therapeutic immunosuppression to temporarily characterize rejection. All animals recovered fully with full active range of motion of the upper extremities the day of surgery and thereafter. Autografts showed no evidence of rejection. Non-immunosuppressed allografts were rapidly rejected showing severe discoloration limited to the allograft and perivenular T cell infiltrate. This was associated with subsequent alloantibody formation and led to graft thrombosis without prominent dermal infiltration. Sub-therapeutically immunosuppressed animals developed a pink/red discoloration slower in progression compared to the unimmunosuppressed animals. These animals also developed alloantibody and rejected in a delayed fashion exhibiting a marked dermal lymphocytic infiltrate similar in magnitude and distribution to previously reported human cases. Specifically, on Hematoxilin and Esosin staining, hyperkeratosis with a seocellular crust and a moderate dermal, perifollicular and perivascular mononuclear cell infiltrate. Moderate multifocal perivascular lymphocytic dermal infiltrates, with mild lymphocytic infiltrates at the dermal-epidermal junction were observed. These lymphoid infiltrates were strongly CD3 positive on immunostaining. Staining with anti-CD68 revealed scattered macrophages in areas of inflammation dominated by the lymphocytes. Anti-C4d stained capillaries weakly in allografts and biopsies of native skin and autografts suggesting that C4d may be a normal finding in these tissues and therefore not related to humoral rejection. Highlights of the histologic findings in other tissues from the immunosuppressed group showed that some animals had mild vasculitis with associated venous thrombosis in the skin. The skeletal muscle had multifocal areas of necrosis with foci of mineralization and regeneration and moderate multifocal perivascular lymphoid infiltrates. Examination of the vessel segments revealed mild to moderate lymphocytic infiltrates around small vessels in adventitia. The sections of the nerve had mild to moderate perivascular infiltrates of lymphocytes with mild infiltrates of lymphocytes within nerve bundles. The inflammatory reaction involving the tendon was generally restricted the blood vessels in the peritendinous connective tissue with mild to moderate perivascular lymphocytic infiltrates. Serum samples obtained from all recipients before transplantation contained no detectable specific alloreactive antibody directed toward donor CD3+ T cells. Recipients without immunosuppression demonstrated detectable alloreactive antibody in their serum following allograft rejection. Immunosuppressed recipients demonstrated detectable alloantibody following allograft rejection, and FACS analysis showed specific binding of alloreactive antibody to donor CD3+ T cells but not autologous T cells. Conclusion. We established a non-human primate model for CTA that is well tolerated by NHPs, results in allosensitization, is responsive to immunosuppression, allows for the evaluation of CTA histology and can be used for the systematic pre-clinical evaluation of therapeutic maneuvers to improve allograft survival.
